Presynaptic Receptor Complexes

描述（由申请人提供）： α-latrotoxin是一种突触前神经毒素，刺激大量突触囊泡胞吐。α-latrotoxin通过与高亲和力神经元细胞表面受体结合而起作用，所述受体被认为代表突触前神经递质释放位点的特异性标记。我们以前的研究鉴定了三种属于不同信号通路的α- latrotoxin受体：CIRLs是G蛋白偶联受体，neurexins是具有单个跨膜结构域和短胞质尾的细胞粘附样蛋白，而PTP 6是受体样蛋白酪氨酸磷酸酶。为了了解α-latrotoxin受体如何耦合突触粘附神经元胞吐，我们建议确定受体相互作用的蛋白质，并研究其相互作用的机制。我们的实验方法是基于使用一种独特的工具，α-latrotoxin，其以高亲和力结合受体，从而允许受体与相关蛋白质一起的一步分离。我们提出了两组实验。第一个是鉴定亲和纯化的α-latrotoxin受体制剂中存在的丰度较低的蛋白质组分，并分析它们与受体相互作用的特异性。这将通过现代蛋白质组学技术，辅以受体特异性和相关蛋白质特异性抗体的免疫沉淀来实现。作为关键阴性对照，将使用从缺乏α-latrotoxin受体的敲除小鼠的脑中获得的受体制剂。第二组实验是研究单个α-latrotoxin受体与突触结合蛋白和突触融合蛋白（syntaxin）的相互作用的机制，所述突触结合蛋白和突触融合蛋白参与结合α-latrotoxin受体的胞吐作用。特别是，我们将确定这些蛋白质与哪些受体结合，并测试这种相互作用是否直接。我们还将确定受体及其相关蛋白质的相互作用结构域。获得a-latrotoxin受体复合物的更好的生物化学定义是重要的，因为它有望提供对参与其功能的信号传导机制的了解，并且可以提供对突触前细胞粘附的了解。这项研究将主要在俄罗斯Shemyakin-Ovchinnikov生物有机化学研究所与亚历山大Petrenko合作进行，作为NIH资助R37 MH 5280的延伸。

DESCRIPTION (provided by applicant): a-Latrotoxin is a presynaptic neurotoxin that stimulates massive synaptic vesicle exocytosis. a-latrotoxin acts by binding to high-affinity neuronal cell-surface receptors that are thought to represent specific markers of presynaptic neurotransmitter release sites. Our previous studies identified three a- latrotoxin receptors that belong to different signaling pathways: The CIRLs are G protein-coupled receptors, neurexins are cell adhesion-like proteins with a single transmembrane domain and short cytoplasmic tail, and PTP6 is a receptor-like protein tyrosine phosphatase. To understand how a-latrotoxin receptors couple synaptic adhesion to neuronal exocytosis, we propose to identify receptor-interacting proteins and to study the mechanisms of their interactions. Our experimental approach is based on the use of a unique tool, a-latrotoxin, that binds the receptors with high affinity, thus allowing the one-stage isolation of the receptors together with associated proteins. We propose two sets of experiments. The first one is to identify less abundant protein components present in the preparations of affinity-purified a-latrotoxin receptors, and to analyze the specificity of their interaction with the receptors. This will be achieved by modern proteomics techniques complemented by immunoprecipitation with receptor-specific and associated protein-specific antibodies. As a key negative control, receptor preparations obtained from brains of knockout mice lacking a-latrotoxin receptors will be used. The second set of experiments is to study the mechanism of the interaction of individual a-latrotoxin receptors with synaptotagmin and syntaxin, proteins, involved in exocytosis that bind to a-latrotoxin receptors. In particular, we will identify to which receptors these proteins bind, and test if this interaction is direct. We will also determine the interacting domains of the receptors and their associated proteins. Obtaining a better biochemical definition of the a-latrotoxin receptor complexes is important because it promises to provide insight into the signaling mechanisms involved in their function, and may provide insight into presynaptic cell adhesion. This research will be performed primarily in Russia at the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry in collaboration with Alexander Petrenko as an extension of NIH grant R37 MH5280.